The long-term goal of this laboratory is reveal the structure of the hair cell's mechanosensitive organelle, the hair bundle, and to determine how structural features of the bundle are responsible for its mechanotransduction function. In this proposal, we extend on the accomplishments of the previous funding period-where we developed a parts list for hair bundles-and here determine the sequence in which bundle proteins are expressed during development, as well as the mechanisms that control their expression and function. In Aim 1, we focus on transcripts for ~175 key bundle proteins, using multiplex RT-qPCR assays to quantify their dynamics in whole sensory epithelium and in pools of isolated hair cells. We also examine the same transcript in isolated single cells, allowing a more detailed understanding of the timing and stochastic nature of gene expression. In Aim 2, we quantify many or most of these proteins by targeted proteomics assays, and also determine their time course for transfer from the soma to the bundle. In Aim 3, we focus on structure; using cryo-electron tomography we will gain a fine understanding of stereocilium structure, while we will use immunofluorescence and structured illumination microscopy to determine which proteins target to which structures. Using conventional conditional alleles or CRISPR/Cas9 knockouts, experiments in both Aims 1 and 3 will test the roles of specific transcription factors and bundle proteins in, respectively, control of bundle transcript expression and bundle structure. We anticipate that some of the bundle proteins we are examining will be affected in genetic diseases that cause hearing impairment.